Western blot

FDB muscles were homogenized using an electric homogenizer (Fluko, Shanghai, China) in a lysis buffer containing in mM: 20Tris-HCl (pH 7.5), 1% Triton X-100, 2 EDTA, 20 NaF, 1 Na₂P₂O₇, 10% glycerol, 150 NaCl, 10 Na₃VO₄, 1 PMSF and protease inhibitors (Complete™, Roche Applied Science). Proteins were separated using SDS-PAGE and transferred to PVDF membranes. The following primary antibodies and their dilutions were used as follows: Total NF-κB total p65 subunit (1:1000; Cell Signaling); p-Ser536-p65 (1:1000; Cell Signaling); p47^phox (1:5000; Sigma); p-p47^phox (pSer359) (1:5000; Sigma); gp91^phox (1:2000; BD Transduction); p-p38 (thr180/tyr182) (1:2000; Santa Cruz); p38 (1:2000; Santa Cruz); Phospho-ERK1/2(Thr202/Tyr204)(1:2000; Cell Signaling); ERK 1/2 (1:2000; Santa Cruz); α-Tubulin (1:5000; Cell Signaling); anti-mouse IgG-HRP (1:20,000; Santa Cruz); anti-rabbit IgG-HRP (1:30,000; Thermo Scientific Pierce). The protein bands in the blots were visualized using a WESTAR Supernova detection kit (Cyanagen, Bologna, Italy) and ChemiDoc™ MP System (Bio-Rad, USA). The intensity of the bands was determined with ImageJ densitometry analysis.